Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2
Abstract
Synthetic methylotrophy, the modification of organisms such as E. coli to grow on methanol, is a longstanding goal of metabolic engineering and synthetic biology. The poor kinetic properties of NAD-dependent methanol dehydrogenase, the first enzyme in most methanol assimilation pathways, limit pathway flux and present a formidable challenge to synthetic methylotrophy. To address this bottleneck, we used a formaldehyde biosensor to develop a phage-assisted noncontinuous evolution (PANCE) selection for variants of Bacillus methanolicus methanol dehydrogenase 2 (Bm Mdh2). Using this selection, we evolved Mdh2 variants with up to 3.5-fold improved Vmax. The mutations responsible for enhanced activity map to the predicted active site region homologous to that of type III iron-dependent alcohol dehydrogenases, suggesting a new critical region for future methanol dehydrogenase engineering strategies. Evolved Mdh2 variants enable twice as much 13C-methanol assimilation into central metabolites than previously reported state-of-the-art methanol dehydrogenases. This work provides improved Mdh2 variants and establishes a laboratory evolution approach for metabolic pathways in bacterial cells.
- Authors:
-
[2];
[1]
- Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States, Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 02138, United States, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States
- Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
- Publication Date:
- Research Org.:
- Harvard Univ., Cambridge, MA (United States)
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 1501961
- Alternate Identifier(s):
- OSTI ID: 1508841
- Grant/Contract Number:
- AR0000433
- Resource Type:
- Published Article
- Journal Name:
- ACS Synthetic Biology
- Additional Journal Information:
- Journal Name: ACS Synthetic Biology Journal Volume: 8 Journal Issue: 4; Journal ID: ISSN 2161-5063
- Publisher:
- American Chemical Society
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; directed evolution; methanol assimilation; methanol dehydrogenase; phage-assisted continuous evolution; phage-assisted noncontinuous evolution; synthetic methylotrophy
Citation Formats
Roth, Timothy B., Woolston, Benjamin M., Stephanopoulos, Gregory, and Liu, David R. Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2. United States: N. p., 2019.
Web. doi:10.1021/acssynbio.8b00481.
Roth, Timothy B., Woolston, Benjamin M., Stephanopoulos, Gregory, & Liu, David R. Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2. United States. https://doi.org/10.1021/acssynbio.8b00481
Roth, Timothy B., Woolston, Benjamin M., Stephanopoulos, Gregory, and Liu, David R. Mon .
"Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2". United States. https://doi.org/10.1021/acssynbio.8b00481.
@article{osti_1501961,
title = {Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2},
author = {Roth, Timothy B. and Woolston, Benjamin M. and Stephanopoulos, Gregory and Liu, David R.},
abstractNote = {Synthetic methylotrophy, the modification of organisms such as E. coli to grow on methanol, is a longstanding goal of metabolic engineering and synthetic biology. The poor kinetic properties of NAD-dependent methanol dehydrogenase, the first enzyme in most methanol assimilation pathways, limit pathway flux and present a formidable challenge to synthetic methylotrophy. To address this bottleneck, we used a formaldehyde biosensor to develop a phage-assisted noncontinuous evolution (PANCE) selection for variants of Bacillus methanolicus methanol dehydrogenase 2 (Bm Mdh2). Using this selection, we evolved Mdh2 variants with up to 3.5-fold improved Vmax. The mutations responsible for enhanced activity map to the predicted active site region homologous to that of type III iron-dependent alcohol dehydrogenases, suggesting a new critical region for future methanol dehydrogenase engineering strategies. Evolved Mdh2 variants enable twice as much 13C-methanol assimilation into central metabolites than previously reported state-of-the-art methanol dehydrogenases. This work provides improved Mdh2 variants and establishes a laboratory evolution approach for metabolic pathways in bacterial cells.},
doi = {10.1021/acssynbio.8b00481},
journal = {ACS Synthetic Biology},
number = 4,
volume = 8,
place = {United States},
year = {Mon Mar 11 00:00:00 EDT 2019},
month = {Mon Mar 11 00:00:00 EDT 2019}
}
https://doi.org/10.1021/acssynbio.8b00481
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Figures / Tables:
Figure 1: Phage assisted noncontinuous evolution of methanol dehydrogenase. (A) General procedure for PANCE. Starting from a saturated, overnight culture of host cells containing the accessory plasmid (AP) and mutagenesis Plasmid (MP), cultures are (1) diluted; (2) grown to logphase; (3) infected with selection phage (SP) and treated with desiredmore »
Works referencing / citing this record:
Continuous evolution of base editors with expanded target compatibility and improved activity
journal, July 2019
- Thuronyi, Benjamin W.; Koblan, Luke W.; Levy, Jonathan M.
- Nature Biotechnology, Vol. 37, Issue 9
Continuous evolution of SpCas9 variants compatible with non-G PAMs
journal, February 2020
- Miller, Shannon M.; Wang, Tina; Randolph, Peyton B.
- Nature Biotechnology, Vol. 38, Issue 4
Growth of E. coli on formate and methanol via the reductive glycine pathway
journal, February 2020
- Kim, Seohyoung; Lindner, Steffen N.; Aslan, Selçuk
- Nature Chemical Biology, Vol. 16, Issue 5
Figures / Tables found in this record: